A commitment has been made in recent years by several governments to support the sequencing of the entire human genome. The human genome contains roughly three billion base pairs of DNA, and consequently the project has generated immense interest in large-scale DNA sequencing. Even after the complete human genome has been sequenced, the determination of individual genotypes in clinical screening remains an important consideration for DNA sequencing. Genotyping performed using techniques such as Southern blotting will suffer from imprecision, and even at best cannot provide detailed sequence information. A simplified method for rapid DNA sequencing would allow routine clinical determination of medically important genes.
Traditional methods for obtaining a DNA sequence all share a fundamental approach. A DNA species is isolated, and a complete nested set of DNA fragments is generated, each fragment having a common starting point and being one base longer than the preceding fragment. The set of fragments is commonly produced by either limited chemical cleavage (Maxam and Gilbert method), or enzymatically, by DNA synthesis in the presence of a small amount of a chain terminating nucleotide (Sanger method). Both methods utilize recognition of the specific bases that comprise DNA to generate sets of fragments, each corresponding to termination at a single base species.
The nested fragments are then size fractionated to determine the order of bases. Polyacrylamide gel electrophoresis has been the method of choice for a number of years, however it suffers from limitations in the length of fragments that can be resolved, and in the length of time required for good separation. In order to determine the nucleotide sequence, the fragments must be labeled with a detectable label, such as a radioisotope or fluorochrome. The label is used to visualize the separation pattern.
The length of sequence data available from a single reaction set as described above is limited to perhaps one thousand bases. The reactions and separations are time-consuming, expensive, and require a skilled technician. Resolution by gel electrophoresis may lead to inaccuracies, and limit the length of sequence which is obtained from a given set of reactions. It is therefore of interest to devise means by which DNA sequencing can be simplified, particularly utilizing methods which lend themselves to automation.